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Figure S1. Assembly of IL-12 and IL-23. (A) IFN-γ–treated or untreated mono-DCs were labeled with [35S]methionine and stimulated with 1 µg/ml LPS. Supernatants were immunoprecipitated after 18 h with a mixture of anti–IL-12 p40 C11.79+C8.6 mAbs (C11+C8; left). IFN-γ–primed cells (right) were immunoprecipitated with anti–IL-12 p35 12H4 mAb, and after three further immunoprecipitation steps with 12H4 mAb, the sample was split into two equal parts and immunoprecipitated with a mixture of anti–IL-23 p19 512+187 mAbs or anti–IL-12 p40 C11+C8 mAbs, and analyzed by SDS-PAGE under nonreducing conditions. Molecular mass is expressed in kilodaltons. Results are representative of three experiments with similar results. (B and C) IL-4–treated, IFN-γ–treated, monocyte-enriched PBMCs (moPBMC) were labeled with [35S]methionine and stimulated with 1 µg/ml LPS. (B) Supernatants (SN) and cell lysates (CL) were immunoprecipitated after 7 h with a mixture of anti–IL-23 p19 512+187 mAbs and resolved in nonreducing SDS-PAGE (left). Bands corresponding to CLp61, CLp57, SNp61, SNp57, and SNp51 were separately excised from the gel, reduced, alkylated, and resolved in SDS-PAGE under reducing conditions (right). CLp51 was poorly resolved in SDS-PAGE, and insufficient material was recovered for SDS-PAGE analysis under reducing conditions. Molecular mass is expressed in kilodaltons. (C) Two-dimensional peptide mapping of IL-12 components and of IL-23 p19. Supernatant was immunoprecipitated with anti–IL-12 p35 12H4 mAb, or after further depletion of residual IL-12 with 12H4 mAb, sequentially immunoprecipitated with anti–IL12 p40 C11+C8 mAbs, and resolved in SDS-PAGE under reducing conditions (not depicted). SNp35 and SNp33 bands derived from 12H4-immunoprecipitated material, corresponding to different glycosylated forms of IL-12 p35, and SNp41, SNp34, and SNp19 bands derived from IL-12 p40 C11+C8–immunoprecipitated material were each excised from the gel, reduced, alkylated, pepsin digested, and resolved by thin layer chromatography with electrophoresis in the first dimension and ascending chromatography in the second dimension.